The present invention provides a novel class of thermally stable and pH stable subtilisin analogs and to a method for preparing such analogs. In particular, the present invention relates to a class of subtilisin analogs having a modified calcium binding site providing improved calcium binding capacity and optionally a deletion and/or replacement of either residue of Asn-Gly sequences present in the subtilisin. The present invention further relates to detergent compositions containing such subtilisins and to the use of such subtilisins and compositions in cleaning applications.
The term subtilisin designates a group of extracellular alkaline serine proteases produced by various species of Bacilli. These enzymes are also referred to as Bacillus serine proteases, Bacillus subtilisins or bacterial alkaline proteases.
Bacillus subtilisin molecules are composed of a single polypeptide chain of either 274 residues (for subtilisin type Carlsberg produced by Bacillus licheniformis and for the subtilisin produced by Bacillus subtilis strain DY) or 275 residues (for subtilisin type BPN' produced by Bacillus amyloliquefaciens, the aprA gene product of Bacillus subtilis, and the subtilisin of Bacillus mesentericus). When comparing amino acid sequences of subtilisin from different strains of Bacillus herein, the sequence of subtilisin BPN' is used as a standard. For example, based on an alignment of sequences that gives the highest degree of homology between subtilisin Carlsberg and subtilisin BPN', the serine at the active site of the former is referred to a serine 221, even though it is located at position 220 of the amino acid sequence. On the same basis, position 220 of the amino acid sequence of subtilisin Carlsberg may be said to "correspond" to position 221 of subtilisin BPN'. See e.g., Nedkov et al., Hoppe-Seyler's Z. Physiol. Chem., 364, 1537-1540 (1983).
The X-ray structure of subtilisin BPN' [Wright, et al., Nature, 221, 235 (1969)] revealed that the geometry of the catalytic site of subtilisin, involving Asp.sup.32, His.sup.64 and Ser.sup.221, is almost identical to that of the active site of mammalian serine proteases (e.g., chymotrypsin) involving the residues Asp.sup.102, His.sup.57, and Ser.sup.195. However, the overall dissimilarities between Bacillus serine proteases and mammalian serine proteases indicate that these are two unrelated families of proteolytic enzymes.
In the family of Bacillus subtilisins complete amino acid sequences are available for five subtilisins: Carlsberg, [Smith, et al., J. Biol. Chem., 243, 2184-2191 (1968)]; BPN' [Markland, et al., J. Biol. Chem., 242, 5198-5211 (1967)]; the aprA gene product [Stahl, et al., J. Bacteriol., 158, 411-418 (1984)]; DY [Nedkov, et al., supra] and Bacillus mesentericus[Svendsen, et al., FEBS Letters, 196, 220-232 (1986)]. Subtilisin Carlsberg and subtilisin BPN' (sometimes referred to as subtilisin Novo) differ by 84 amino acids and one additional residue in BPN' (subtilisin Carlsberg lacks an amino acid residue corresponding to residue 56 of subtilisin BPN'). Subtilisin DY comprises 274 amino acids and differs from subtilisin Carlsberg in 32 amino acid positions and from subtilisin BPN' by 82 amino acid replacements and one deletion (subtilisin DY lacks an amino acid residue corresponding to residue 56 of subtilisin BPN'). The amino acid sequence of the aprA gene product is 85% homologous to the amino acid sequence of subtilisin BPN'. Thus, it appears that there is an extensive homology between amino acid sequences of subtilisins from different strains of Bacillus. This homology is complete in certain regions of the molecule and especially in those that play a role in the catalytic mechanism and in substrate binding. Examples of such sequence invariances are the primary and secondary substrate binding sites, Ser.sup.125 -Leu.sup.126 -Gly.sup.127 -Gly.sup.128 and Tyr.sup.104 respectively and the sequence around the reactive serine (221), Asn.sup.218 -Gly.sup.219 -Thr.sup.220 -Ser.sup.221 -Met.sup.222 -Ala.sup.223.
Subtilisin molecules exhibit unique stability properties. Although they are not completely stable over a wide pH range, subtilisins are relatively resistant to denaturation by urea and guanidine solutions and their enzymatic activity is retained for some time in 8M urea. In solutions having a pH below 4, subtilisin rapidly and irreversibly loses its proteolytic activity. Gounaris, et al., Compt. Rend. Trav. Lab. Carlsberg, 35, 37 (1965) demonstrated that the acid deactivation of subtilisin is not due to a general charge effect and speculated that it is due to other changes in the molecule, such as protonation of histidine residues in the interior, hydrophobic parts of the molecule. Bacillus subtilisins undergo irreversible inactivation in aqueous solutions at a rate that is largely dependent upon temperature and pH. At pH values below 4 or above 11 the rate of inactivation is very rapid while at pH's of between 4.5 and 10.5 the rate, although much slower, increases as the solution becomes more alkaline. The mechanisms of this inactivation are not fully known but there is evidence indicating that autodigestion is responsible at least in part for enzyme instability at this pH range. In general, at any pH value, the higher the temperature the faster the rate of subtilisin deactivation.
The use of proteases in industrial processes which require hydrolysis of proteins has been limited due to enzyme instability under operational conditions. Thus, for example, the incorporation of trypsin into laundry detergents (e.g., Bio-38, Schnyder; Switzerland) to facilitate removal of proteinaceous stains had a very limited success which was undoubtedly a result of enzyme instability under the washing conditions. In addition, bacterial alkaline proteases compatible with detergents have been utilized in detergent formulations.
Because many industrial processes are conducted at temperatures that are above the stability range of most enzymes, highly thermostable proteases not only will be advantageous to certain industries such as detergent and hide dehairing, that already require stable proteases, but may be useful in industries that use chemical means to hydrolyze proteins e.g. hydrolysis of vegetable and animal proteins for the production of soup concentrates.
Although thermal inactivation may be the most important factor in restricting the industrial use of enzymes, other factors such as need for effectiveness over broad pH ranges and use of denaturing agents may also have a detrimental effect with respect to the use of proteases in industrial processes. It is therefore desirable to obtain a class of proteases characterized by improved stability with respect to temperature, pH, denaturing agents and other conditions required by various industries.
Over the past several years there have been major changes in detergent formulations, particularly in the replacement of phosphates with alternate builders and in the development of liquid laundry detergents to meet environmental and consumer demands. These changes create a need for changes in traditional detergent enzymes. More particularly, it has become desirable to employ proteolytic enzymes which possess greater storage stability in liquid laundry formulations as well as stability and activity at broader ranges of pH and temperature.
One approach to producing modified subtilisins useful in detergent formulations was disclosed in European patent application No. 130,756, wherein mutations in the subtilisin of Bacillus amyloliquefaciens (B. amyloliquefacines) at positions Tyr.sup.-1, Asp.sup.32, Asn.sup.155, Tyr.sup.104, Met.sup.222, Gly.sup.166, His.sup.64, Gly.sup.169, Phe.sup.189, Ser.sup.33, Ser.sup.221, Tyr.sup.217, Glu.sup.156, and/or Ala.sup.152 were identified as providing changed stability, altered conformation or as having changes in the "processing" of the enzyme. In particular, a mutation of Met.sup.222 to Ala or Cys (which mutant also exhibits a sharper pH optimum than wild type) or Ser assertedly resulted in improved oxidation stability. It was suggested that substitution for Gly.sup.166 with Ala, Asp, Glu, Phe, His, Lys, Asn, Arg or Val would alter the kinetic parameters of the enzyme. However, none of the mutations disclosed provide analogs having greater stability at high temperatures or stability over a broader pH range than the wild type enzyme.
In another approach, Thomas, et al, Nature 318, 375-376 (1985), disclosed that the pH dependence of subtilisin may be altered by changing an Asp to Ser in Asp.sup.99 -Gly.sup.100 of subtilisin BPN'. This change represents an alteration of a surface charge 14-15 Angstroms from the active site. However, Thomas, et al. fails to provide any indication of improvement where no change in surface charge is made, as is the case where one uncharged residue is substituted for another.
A third approach, described in co-pending U.S. application Ser. No. 819,241 now abandoned, relates to a class of Bacillus serine protease analogs characterized by deletion and/or modifications of any Asn-Gly sequences present in the protease.